Analytical strategies for the comprehensive profiling of histone post translational modifications by mass spectrometry and implications for functional analyses

Le long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi.

Macrocicles nitrogenats contenint unitats de ferrocè i llurs complexos de Pal·ladi(0). Estudi estructural i aplicacions en catàlisi

La present tesi doctoral s'ha basat en la caracterització estructural i en l'estudi de les propietats catalítiques de nous complexos de pal·ladi(0) amb lligands olefínics contenint unitats de ferrocè. En una primera fase s'ha desenvolupat la síntesi d'estructures de tipus (E, E, E)-1,6,11-trisarilsulfonil-1,6,11-triazaciclopentadeca-3,8,13-triè contenint des d'una fins a tres unitats de ferrocè i s'ha estudiat la seva complexació amb pal·ladi(0). Mitjançant estudis electroquímics s'ha avaluat la seva possible aplicació en el reconeixement molecular electroquímic. Encara que aquest tipus d'estructures no han resultat vàlides per aquest tipus de processos de reconeixement, l'estudi electroquímic ha permès determinar que la presència del grup ferrocenil influeix en les propietats redox del metall complexat. S'ha descrit també l'obtenció de macrocicles pentaolefínics anàlegs de 25 membres contenint unitats de ferrocè i s'ha estudiat paral·lelament la seva complexació amb pal·ladi(0).En una segona part del treball s'han avaluat les propietats catalítiques del complex (E, E, E)-6,11-bis[(p-metilfenil)sulfonil]-1-ferrocenilsulfonil-1,6,11-triazaciclopentadeca-3,8,13-trièpal·ladi(0), en reaccions clàssicament catalitzades per pal·ladi tals com l'acoblament creuat de Suzuki o la reacció de Heck, i s'ha pogut determinar que és un catalitzador actiu en aquest tipus de reaccions, essent a més possible, la seva recuperació i reutilització. En el cas concret de la reacció de Heck s'han emprat sals de diazoni com a agents arilants i cal destacar que s'ha descrit el primer sistema catalític recuperable i reutilitzable per aquest tipus de reaccions. Mitjançant espectrometria de masses amb ionització per electrospray ha estat possible determinar el rol mecanístic del nostre complex de pal·ladi en la reacció de Heck amb sals de diazoni.Finalment, en una tercera part estructural del treball s'ha estudiat el comportament espectroscòpic dels complexos de pal·ladi(0) anteriorment mencionats, contenint des de tres unitats aríliques iguals fins a tres unitats aríliques diferents. Mitjançant RMN i difracció de raigs-X s'ha determinat que la complexitat estereoquímica dels complexos deriva dels isòmers que es poden formar mitjançant complexació del metall amb cada una de les cares dels tres dobles enllaços continguts a l'estructura.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye (TM) and analysed by two-dimensional difference gel. electrophoresis. Gel images analysed off-line, using the DeCyder (TM) image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of X (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data. suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens. (c) 2009 Elsevier Ltd. All rights reserved.

Analysis of residues of prometryne and acetochlor in soil-water system by solid-phase extraction and gas chromatography/mass spectrometry

A highly sensitive and simple analytical method was developed for analyzing the binary mixed pesticides of prometryne and acetochlor in soil–water system by gas chromatography/mass spectrometry (GC/MS). The sample solution was first purified by C18 solid-phase extraction column, which was leached by acetone. The leachate was enriched to 1.0 mL by pressure blowing concentrator and then analyzed by GC/MS. The linear calibration curves were showed in the range of 1–15 μg/mL with a correlation coefficient of 0.9991. The average recoveries (n = 5) were between 95.3 and 115.7%, with relative standard deviations ranged from 1.71 and 7.95%. The limits of detection of Prometryne/Acetochlor were up to 0.06 and 0.17 μg/mL, respectively. This method provides a reliable approach to examine and evaluate the residues of prometryne and acetochlor in the soil–water system.

Quantification of sex pheromone from Anocentor nitens by gas chromatography-mass spectrometry-selected ion monitoring

Two highly sensitive and selective methods based on gas chromatography coupled to mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode have been developed for the quantification of 2,6-dichlorophenol (2,6-DCP), a sex pheromone of the tick females of Anocentor nitens. Standard addition method and calibration curve techniques using 5-bromine-4-hydroxy-3- methoxybenzaldehyde (5-BrV) as internal standard (IS) afforded detection limit of 0.1ngml-1. The calibration curve was linear over the concentration range from 0.5 to 500ngml-1 for 2,6-DCP. Results show that the concentration range of sex pheromone in the extracts samples was 1.08-10.35ngml-1. The methods developed provided reliable procedures to determine amounts of 2,6-DCP present in ticks. © 2003 Elsevier B.V. All rights reserved.

Peptide diversity in the venom of the social wasp Polybia paulista (Hymenoptera): A comparison of the intra- and inter-colony compositions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of rocuronium in human plasma by liquid chromatography-tandem mass spectrometry with application in clinical pharmacokinetics

Rocuronium (ROC) is a neuromuscular blocking agent used in surgical procedures which is eliminated primarily by biliary excretion. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for analysis of ROC in human plasma. Separation of ROC and IS (verapamil) was performed using an endcapped C-18 column and a mixture of water:acetonitrile:trifluoracetic acid (50:50:0.1, v/v) as mobile phase. Aliquots of 100 mu L of human plasma were extracted at pH 3, using dichloromethane. The lower limit of quantification of 5 ng/mL shows the high sensitivity of this method. Intra- and inter-assay precision (as relative standard deviation) was all <= 14.2% and accuracy (as relative standard error) did not exceed 10.1%. The validated method was successfully applied to quantify ROC concentrations in patients under surgical procedures up to 6 h after the administration of the 0.4-0.9 mg/kg ROC. The pharmacokinetic parameter estimations of ROC showed AUC/dose of 563 mu g min/mL, total clearance of 2.5 mL/min/kg, volume of distribution at steady state of 190 mL/kg and mean residence time of 83 min. (C) 2013 Elsevier B.V. All rights reserved.

Abordagem metalômica quantitativa de mercúrio em peixes da região amazônica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mercury fractionation in dourada (Brachyplatystoma rousseauxii) of the Madeira River in Brazil using metalloproteomic strategies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of trace elements in bovine semen samples by inductively coupled plasma mass spectrometry and data mining techniques for identification of bovine class

The reproductive performance of cattle may be influenced by several factors, but mineral imbalances are crucial in terms of direct effects on reproduction. Several studies have shown that elements such as calcium, copper, iron, magnesium, selenium, and zinc are essential for reproduction and can prevent oxidative stress. However, toxic elements such as lead, nickel, and arsenic can have adverse effects on reproduction. In this paper, we applied a simple and fast method of multi-element analysis to bovine semen samples from Zebu and European classes used in reproduction programs and artificial insemination. Samples were analyzed by inductively coupled plasma spectrometry (ICP-MS) using aqueous medium calibration and the samples were diluted in a proportion of 1:50 in a solution containing 0.01% (vol/vol) Triton X-100 and 0.5% (vol/vol) nitric acid. Rhodium, iridium, and yttrium were used as the internal standards for ICP-MS analysis. To develop a reliable method of tracing the class of bovine semen, we used data mining techniques that make it possible to classify unknown samples after checking the differentiation of known-class samples. Based on the determination of 15 elements in 41 samples of bovine semen, 3 machine-learning tools for classification were applied to determine cattle class. Our results demonstrate the potential of support vector machine (SVM), multilayer perceptron (MLP), and random forest (RF) chemometric tools to identify cattle class. Moreover, the selection tools made it possible to reduce the number of chemical elements needed from 15 to just 8.

Multi-element determination in Brazilian honey samples by inductively coupled plasma mass spectrometry and estimation of geographic origin with data mining techniques

Multi-element analysis of honey samples was carried out with the aim of developing a reliable method of tracing the origin of honey. Forty-two chemical elements were determined (Al, Cu, Pb, Zn, Mn, Cd, Tl, Co, Ni, Rb, Ba, Be, Bi, U, V, Fe, Pt, Pd, Te, Hf, Mo, Sn, Sb, P, La, Mg, I, Sm, Tb, Dy, Sd, Th, Pr, Nd, Tm, Yb, Lu, Gd, Ho, Er, Ce, Cr) by inductively coupled plasma mass spectrometry (ICP-MS). Then, three machine learning tools for classification and two for attribute selection were applied in order to prove that it is possible to use data mining tools to find the region where honey originated. Our results clearly demonstrate the potential of Support Vector Machine (SVM), Multilayer Perceptron (MLP) and Random Forest (RF) chemometric tools for honey origin identification. Moreover, the selection tools allowed a reduction from 42 trace element concentrations to only 5. (C) 2012 Elsevier Ltd. All rights reserved.

Microextraction techniques coupled to liquid chromatography with mass spectrometry for the determination of organic micropollutants in environmental water samples

[EN]Until recently, sample preparation was carried out using traditional techniques, such as liquid–liquid extraction (LLE), that use large volumes of organic solvents. Solid-phase extraction (SPE) uses much less solvent than LLE, although the volume can still be significant. These preparation methods are expensive, time-consuming and environmentally unfriendly. Recently, a great effort has been made to develop new analytical methodologies able to perform direct analyses using miniaturised equipment, thereby achieving high enrichment factors, minimising solvent consumption and reducing waste. These microextraction techniques improve the performance during sample preparation, particularly in complex water environmental samples, such as wastewaters, surface and ground waters, tap waters, sea and river waters. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) and time-of-flight mass spectrometric (TOF/MS) techniques can be used when analysing a broad range of organic micropollutants. Before separating and detecting these compounds in environmental samples, the target analytes must be extracted and pre-concentrated to make them detectable. In this work, we review the most recent applications of microextraction preparation techniques in different water environmental matrices to determine organic micropollutants: solid-phase microextraction SPME, in-tube solid-phase microextraction (IT-SPME), stir bar sorptive extraction (SBSE) and liquid-phase microextraction (LPME). Several groups of compounds are considered organic micropollutants because these are being released continuously into the environment. Many of these compounds are considered emerging contaminants. These analytes are generally compounds that are not covered by the existing regulations and are now detected more frequently in different environmental compartments. Pharmaceuticals, surfactants, personal care products and other chemicals are considered micropollutants. These compounds must be monitored because, although they are detected in low concentrations, they might be harmful toward ecosystems.

Characterization of circulating steroids and endocannabinoids levels in obese subjects by liquid chromatography tandem mass spectrometry

Nowadays obesity can be defined as a global epidemic. The precise identification of circulating biomarkers involved in this pathology could be essential to early diagnose potential co-morbidities and to better address the development of future therapeutic strategies. Published evidences show that circulating steroid hormones and endocannabinoids might have a role in the physiopathology of obesity; however, a precise and reliable quantification of these molecules is still lacking. In the first part of the present thesis, we developed a sensitive, specific and accurate quantification method for nine steroid hormones using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system. This method has been used first for a comparative study with immunoassays, currently used in the clinical practice to quantify these molecules and then to redefine circulating reference intervals in healthy subjects. Furthermore, we measured circulating steroid hormones in three groups of subjects: normo-weight, over-weight and obese, defining different steroid hormones profiles depending on the obesity state. The role of circulating endocannabinoids in humans is still unclear, however there are several evidences concerning their involvement in obesity. In the second part of the thesis, we determined changes of circulating endocannabinoids in obese patients after a weight loss induced by bariatric surgery, currently the most effective long-term treatment for obesity, using LC/MS-MS. We measured basal and dynamic endocannabinoids plasma levels in 12 patients with severe obesity before, one month after and six months after the Roux-en-Y gastric bypass intervention, currently one of the most performed types of bariatric surgery. All together the findings illustrated in this thesis project will help better define the role of steroid hormones and endocannabinoids in the framework of obesity in humans and the role that each type of molecule might have in its pathophysiology.